Purification, Cloning and Immuno-Biochemical Characterization of a Fungal Aspartic Protease Allergen Rhi o 1 from the Airborne Mold Rhizopus oryzae

Abstract

BackgroundFungal allergy is considered as serious health problem worldwide and is increasing at an alarming rate in the industrialized areas. Rhizopus oyzae is a ubiquitously present airborne pathogenic mold and an important source of inhalant allergens for the atopic population of India. Here, we report the biochemical and immunological features of its 44 kDa sero-reactive aspartic protease allergen, which is given the official designation ‘Rhi o 1’.MethodThe natural Rhi o 1 was purified by sequential column chromatography and its amino acid sequence was determined by mass spectrometry and N-terminal sequencing. Based on its amino acid sequence, the cDNA sequence was identified, cloned and expressed to produce recombinant Rhi o 1. The allergenic activity of rRhi o 1 was assessed by means of its IgE reactivity and histamine release ability. The biochemical property of Rhi o 1 was studied by enzyme assay. IgE-inhibition experiments were performed to identify its cross-reactivity with the German cockroach aspartic protease allergen Bla g 2. For precise characterization of the cross-reactive epitope, we used anti-Bla g 2 monoclonal antibodies for their antigenic specificity towards Rhi o 1. A homology based model of Rhi o 1 was built and mapping of the cross-reactive conformational epitope was done using certain in silico structural studies.ResultsThe purified natural nRhi o 1 was identified as an endopeptidase. The full length allergen cDNA was expressed and purified as recombinant rRhi o 1. Purified rRhi o 1 displayed complete allergenicity similar to the native nRhi o 1. It was recognized by the serum IgE of the selected mold allergy patients and efficiently induced histamine release from the sensitized PBMC cells. This allergen was identified as an active aspartic protease functional in low pH. The Rhi o 1 showed cross reactivity with the cockroach allergen Bla g 2, as it can inhibit IgE binding to rBla g 2 up to certain level. The rBla g 2 was also found to cross-stimulate histamine release from the effector cells sensitized with anti-Rhi o 1 serum IgE. This cross-reactivity was found to be mediated by a common mAb4C3 recognizable conformational epitope. Bioinformatic studies revealed high degree of structural resemblances between the 4C3 binding sites of both the allergens.Conclusion/SignificanceThe present study reports for the first time anew fungal aspartic protease allergen designated as Rhi o 1, which triggers IgE-mediated sensitization leading to various allergic diseases. Here we have characterized the recombinant Rhi o 1 and its immunological features including cross-reactive epitope information that will facilitate the component-resolved diagnosis of mold allergy.

Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant

“…As a first step, mapping of the IgE epitope and its critical residues was performed. We observed the low degree of immunological relevance of an earlier reported (14) cross-reactive IgE epitope shared by Rhi o 1 and the cockroach allergen Bla g 2. Instead, we identified two novel IgE-binding peptides that together constitute a major epitope of Rhi o 1.…”

“…The mAb4C3 was originally developed against a conformational IgE epitope of Bla g 2. Interestingly, it was observed in our earlier study (14) that Rhi o 1 also had a 4C3 binding site, and this site overlapped with a conformational IgE epitope of this allergen. In the present study, the 4C3 binding site remained intact because there was no mutation induced on it.…”

“…Recombinant Allergens and Monoclonal Antibody-Recombinant Rhi o 1 was expressed in E. coli BL21(DE3)Rosetta and purified using an Ni-NTA column (Qiagen), as described earlier (14). rBet v 1 (an unrelated pollen allergen for negative control) was from Dr. Margarete Focke-Tejkl (Medical University of Vienna).…”

“…In India, emerging clinical and immunological studies have suggested the involvement of Rhizopus oryzae allergens in type I allergy (11)(12)(13). Rhi o 1 is a major allergen (aspartic protease, molecular mass 44 kDa) of R. oryzae, which causes respiratory sensitization to mold allergic patients (14). This allergen has been officially recognized and listed in the database of the International Union of Immunological Societies.…”

“…oryzae-sensitized Patients-Co-sensitization to multiple allergens having conserved molecular surfaces can often give rise to unwanted cross-reactivity of low clinical importance. Here, we studied the significance of an earlier reported (14) cross-reactivity between rRhi o 1 and rBla g 2 by using sera from a population of 45 R. oryzae-sensitized patients. As observed in an IgE dot blot (Fig.…”